Epithelial-to-mesenchymal transition and resistance to ingenol 3-angelate, a novel protein kinase C modulator, in colon cancer cells.

Acquired resistance to protein kinase C (PKC) modulators may explain the failure of clinical trials in patients with cancer. Herein, we established a human colon cancer cell line resistant to PEP005, a drug that inhibits PKCalpha and activates PKCdelta. Colo205-R cells, selected by stepwise exposure to PEP005, were >300-fold more resistant to PEP005 than parental Colo205-S cells and were cross-resistant to phorbol 12-myristate 13-acetate, bryostatin, bistratene A, and staurosporine. No PKCalpha or PKCdelta mutation was detected in Colo205-S and Colo205-R cells. Changes in Colo205-R cells were reminiscent of the epithelial-to-mesenchymal transition (EMT) phenotype. Accordingly, Colo205-R cells were more invasive than Colo205-S in Matrigel assays and in mouse xenografts. We also found an increased mRNA expression of several EMT genes, such as those encoding for transforming growth factor-beta and vimentin, along with a decreased mRNA expression of genes involved in epithelial differentiation, such as CDH1 (E-cadherin), CLDN4 (claudin 4), S100A4, and MUC1, in Colo205-R compared with Colo205-S cells in vitro and in vivo. Interestingly, high expression of ET-1 was shown in Colo205-R cells and correlated with low sensitivity to PEP005 and staurosporine in a panel of 10 human cancer cell lines. Inhibition of the ET-1 receptor ETR-A with bosentan restored the antiproliferative effects of PEP005 in Colo205-R cells and decreased the invasive properties of this cell line. Exogenous exposure to ET-1 and silencing ET-1 expression using small interfering RNA modulated cell signaling in Colo205-S and Colo205-R. In summary, acquired resistance to PEP005 was associated with expression of EMT markers and activates the ET-1/ETR-A cell signaling.

Effects of protein kinase C modulation by PEP005, a novel ingenol angelate, on mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling in cancer cells.

PEP005 (ingenol-3-angelate) is a novel anticancer agent extracted from Euphorbia peplus that was previously shown to modulate protein kinase C (PKC), resulting in antiproliferative and proapoptotic effects in several human cancer cell lines. In Colo205 colon cancer cells, exposure to PEP005 induced a time- and concentration-dependent decrease of cells in S phase of cell cycle and apoptosis. In Colo205 cells exposed to PEP005, a variety of signaling pathways were activated as shown by increased phosphorylation of PKCdelta, Raf1, extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase, p38 MAPK, and PTEN. PEP005-induced activation of PKCdelta was associated with its translocation from the cytosol to the nucleus and other cellular membranes. Interestingly, PEP005 treatment also resulted in reduced expression of PKCalpha and reduced levels of phosphorylated active form of AKT/protein kinase B. These data suggest that PEP005-induced activation of PKCdelta and reduced expression of PKCalpha resulted in apoptosis by mechanisms mediated by activation of Ras/Raf/MAPK and inhibition of the phosphatidylinositol 3-kinase/AKT signaling pathways. This study supports ongoing efforts targeting PKC isoforms in cancer therapy with PEP005 alone and in combination with other cytotoxic agents.

Alterations of the IKBKG locus and diseases: an update and a report of 13 novel mutations.

Mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential modulator (NEMO), gene are the most common single cause of incontinentia pigmenti (IP) in females and anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males. The IKBKG gene, located in the Xq28 chromosomal region, encodes for the regulatory subunit of the inhibitor of kappaB (IkB) kinase (IKK) complex required for the activation of the NF-kB pathway. Therefore, the remarkably heterogeneous and often severe clinical presentation reported in IP is due to the pleiotropic role of this signaling transcription pathway. A recurrent exon 4_10 genomic rearrangement in the IKBKG gene accounts for 60 to 80% of IP-causing mutations. Besides the IKBKG rearrangement found in IP females (which is lethal in males), a total of 69 different small mutations (missense, frameshift, nonsense, and splice-site mutations) have been reported, including 13 novel ones in this work. The updated distribution of all the IP- and EDA-ID-causing mutations along the IKBKG gene highlights a secondary hotspot mutation in exon 10, which contains only 11% of the protein. Furthermore, familial inheritance analysis revealed an unexpectedly high incidence of sporadic cases (>65%). The sum of the observations can aid both in determining the molecular basis of IP and EDA-ID allelic diseases, and in genetic counseling in affected families.

Antiproliferative effects of rapamycin as a single agent and in combination with carboplatin and paclitaxel in head and neck cancer cell lines.

PURPOSE: Recent data suggested that combining targeted therapies with chemotherapy may counteract drug resistance. Activation of the PI3K/AKT/mTOR pathway downstream to kinase receptors, such as EGFR, was found in 57-81% of head and neck squamous cell carcinoma (HNSCC), and was eventually associated with a loss of PTEN function. mTOR was shown to modulate cell proliferation, apoptosis, invasion, and angiogenesis. This study aimed to evaluate molecular and cellular effects of rapamycin in a panel of cell lines either as single agent or in combination with cytotoxics commonly used in HNSCC. METHODS: Antiproliferative effects of rapamycin, carboplatin, and paclitaxel were evaluated in a panel of three HNSCC cell lines (SCC61, SQ20B and HEP2). Cells were exposed to rapamycin for 48 h, to carboplatin for 48 h, or to paclitaxel for 24 h. Antiproliferative effects of simultaneous and sequential rapamycin-based combinations were studied using MTT assay and median effect plot analysis. Cell cycle effects were analysed using flow cytometry. RESULTS: Rapamycin induced concentration dependent antiproliferative effects in HNSCC cell lines with IC(50) of 5 +/- 1, 12 +/- 2 and 20 +/- 2 microM in SCC61, SQ20B, and HEP2 cells, respectively. Higher antiproliferative effects were observed in SCC61 cells overexpressing NOXA and cyclin D1 than in HEP2 that overexpressed MDR1 and BCL2. In our panel, antiproliferative effects of rapamycin were associated with G0/G1 cell cycle accumulation and apoptosis induction, at concentrations ranging 3-30 microM. Combinations of rapamycin with paclitaxel and carboplatin displayed synergistic and additive effects. Synergistic effects were observed with paclitaxel in SQ20B and HEP2 cells and with carboplatin in SQ20B cells, when cells were exposed to cytotoxics prior to rapamycin. CONCLUSION: Our results show that rapamycin displays antiproliferative effects and induces apoptosis in HNSCC cell lines, cellular effects being more potent in cells that do not express BCL2 and MDR1. Additive and synergistic effects were observed when rapamycin was combined with carboplatin and paclitaxel.

Preclinical and clinical development of novel agents that target the protein kinase C family.

Protein kinase C (PKC) family comprises more than 12 serine-/threonine-specific isoenzymes. PKC isoenzymes play a complex role in the transduction of signal from tyrosine kinase receptor modulating proliferation, cell cycle, apoptosis, invasion, differentiation, and senescence in both cancer cells and endothelial cells. Thereby, inhibition and/or activation of specific PKCs is thought to control tumor growth by interacting directly with cancer cells and indirectly by blocking tumor angiogenesis. Furthermore, PKCs are known to modulate multi-drug resistance, providing a rational for the combination of PKCs modulators with classical cytotoxic drugs. During the past few years, preclinical and clinical data with first-generation PKC inhibitors/activators provided insight that PKCs may indeed represent attractive targets for the discovery of small molecules with new anticancer properties. In this review, we will provide an overview on the current understanding of PKC participation in chemotherapeutic resistance, the possible implications in cancer treatment, and the potential of most recent PKC inhibitors in molecular cancer therapeutics.